Tuberculosis kills about 2 million people globally every year. A key defense against Mycobacterium tuberculosis (Mtb) infections is the production of nitric oxide (NO) by macrophages. Although NO controls Mtb growth, it rarely sterilizes the bacterium from the host. Therefore, it is likely that Mtb has mechanisms to resist NO toxicity. Two genes were identified in Mtb, mpa (Mycobacterium proteasome ATPase) and pafA (proteasome associated factor), to be required for protection against NO. Importantly, mpa and pafA mutants are severely attenuated in mice. The proteasome is a multi-subunit, barrel shaped complex that degrades proteins. We hypothesize that Mpa and PafA chaperone proteins into the Mtb proteasome for degradation, but we do not know why this activity protects Mtb against NO or promotes virulence in mice. Thus, the goals of this proposal are to understand why proteasome activity protects Mtb against NO toxicity and promotes bacterial growth in vivo. We will examine proteasome-dependent gene expression to determine if genes regulated by the proteasome are required for NO-resistance or pathogenesis. In addition, we will determine how genes are regulated by the proteasome. Finally, we will identify substrates of the proteasome, which may reveal why protein degradation is critical for resistance to NO and survival in vivo. Relevance to public health: With increased age expectancy, prevalence of HIV infections and number of persons taking immunosuppressive drugs, the chances of contracting tuberculosis increases. Furthermore, tuberculosis therapy takes 6-9 months, a problem that leads to decreased compliance for taking antibiotics and increased chances of developing drug-resistant strains of Mtb. Taken together it will be important to develop faster acting drugs to new targets in Mtb in order to better treat tuberculosis in the future.